In vitro and in silico PTP-1B inhibition and in vivo antidiabetic activity of semisynthetic moronic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2016.02.082

Six derivatives (1-6) of moronic acid were semi-synthesized and their in vitro protein tyrosine phosphatase 1B (PTP-1B) inhibition activity assessed. Derivatives 2 (IC₅₀ = 10.8 ± 0.5 μM) and 6 (IC₅₀ = 7.5 ± 0.1 μM) displayed the most potent inhibitory activity. Therefore, they (50 mg/Kg) were tested for their antidiabetic effect in vivo using a non-insulin dependent diabetes mellitus rat model. The results indicated that they decrease plasma glucose levels during all the experiment (p <0.05). Docking analysis of 2 and 6 with PTP-1B orthosteric site A and allosteric site B, showed that 2 had polar and Van der Waals interactions in both sites with Val49, Gln262, Met258, Phe182, Ala217, Ile219 and Gly259, displaying more affinity for site A. Compound 6 showed polar interaction with Gln262 and Van der Waals with Val49, Ile219, Gly259, Arg254, Ala27, Phe52, Met258, Asp48 and Phe182, suggesting that the potential binding site is localized in site B, close to the catalytic site A. Therefore, derivatives 2 and 6 have potential for the development of antidiabetic agents.

Full text of DOI:10.1016/j.bmcl.2016.02.082

Accepted Manuscript
In vitro and in silico PTP-1B inhibition and in vivo antidiabetic activity of sem-
isynthetic moronic acid derivatives
Litzia Cerón-Romero, Paolo Paoli, Guido Camici, Virginia Flores-Morales,
María Yolanda Rios, Juan J. Ramírez-Espinosa, Sergio Hidalgo-Figueroa,
Gabriel Navarrete-Vázquez, Samuel Estrada-Soto
PII:
S0960-894X(16)30207-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.082
BMCL 23636
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
6 January 2016
25 February 2016
27 February 2016
Please cite this article as: Cerón-Romero, L., Paoli, P., Camici, G., Flores-Morales, V., Rios, M.Y., Ramírez-
Espinosa, J.J., Hidalgo-Figueroa, S., Navarrete-Vázquez, G., Estrada-Soto, S., In vitro and in silico PTP-1B
inhibition and in vivo antidiabetic activity of semisynthetic moronic acid derivatives, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.02.082
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Bioorganic and Medicinal Chemistry Letters
In vitro and in silico PTP-1B inhibition and in vivo antidiabetic
activity of semisynthetic moronic acid derivatives
a
b
b
c
Litzia Cerón-Romero, Paolo Paoli, Guido Camici, Virginia Flores-Morales, María
d
a
a
Yolanda Rios, Juan J. Ramírez-Espinosa, Sergio Hidalgo-Figueroa, Gabriel Navarrete-
Vázquez, Samuel Estrada-Soto
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa, 62209, Cuernavaca,
Morelos, México
Dipartimento di Scienze Biomediche Sperimentali e Cliniche “Mario Serio”, Sezione di Scienze Biochimiche, Universitá degli Studi di
a
a,*
a
b
Firenze, Viale Morgani 50, 50134 Firenze, Italy.
c
Unidad Académica de Ciencias Químicas (LSAyB), Universidad Autónoma de Zacatecas Campus Siglo XXI, Carretera Zacatecas-
Guadalajara, Km. 6.0, Ejido la Escondida, 98160 Zacatecas, México
d
Centro de Investigaciones Químicas, IICBA, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa,
6
2209, Cuernavaca, Morelos, México
ARTICLE INFO
ABSTRACT
Article history:
Received
Six derivatives (1-6) of moronic acid were semi-synthesized and their in vitro protein
tyrosine phosphatase 1B (PTP-1B) inhibition activity assessed. Derivatives 2 (IC = 10.8 ±
5
0
Received in revised form
Accepted
Available online
0.5 M) and 6 (IC = 7.5 ± 0.1 M) displayed the most potent inhibitory activity.
50
Therefore, they (50 mg/Kg) were tested for their antidiabetic effect in vivo using a non-
insulin dependent diabetes mellitus rat model. The results indicated that they decrease
plasma glucose levels during all the experiment (p < 0.05). Docking analysis of 2 and 6
with PTP-1B orthosteric site A and allosteric site B, showed that 2 had polar and Van der
Waals interactions in both sites with Val49, Gln262, Met258, Phe182, Ala217, Ile219 and
Gly259, displaying more affinity for site A. Compound 6 showed polar interaction with
Gln262 and Van der Waals with Val49, Ile219, Gly259, Arg254, Ala27, Phe52, Met258,
Asp48 and Phe182, suggesting that the potential binding site is localized in site B, close to
the catalytic site A. Therefore, derivatives 2 and 6 have potential for the development of
antidiabetic agents.
Keywords:
Diabetes
Moronic acid derivatives
PTP-1B
Molecular docking
Enzyme inhibition
©
2015 Elsevier Ltd. All rights reserved.
Type 2 Diabetes (T2D) is a chronic progressive
The study of medicinal plants and their
compounds has led to the discovery of new chemical
entities for potential development as drugs that act over
disease caused by metabolic deregulation characterized by
insulin resistance in peripheral tissues (liver, muscle and
adipose tissue) and impaired insulin secretion by the
7
new or known therapeutic targets. Moreover, SAR studies
1,2,3
pancreas,
and has been placed at the top of morbidity
of semisynthetic derivatives from a bioactive natural
product are a valuable tool to obtain potent, efficient and
and mortality around the world. Currently, there are an
estimated of 387 million people with diabetes worldwide,
8
low toxicity entities with antidiabetic activity. In this
and it is expected to increase to 592 million of cases in
context, Moronic acid (MA), a natural triterpene obtained
from Phoradendron reichenbachianum, has become an
alternative for the treatment of T2D, due to having
4
2035. Despite lifestyle modification as first-line approach
for early stage diabetic patients, T2D treatment through
drug prescription remains to be the major successfully
strategy to improve this condition. Thus, there are several
7,9,10
pharmacological
properties,
emphasizing
the
antidiabetic activity mediated by the inhibition of protein
5
7
classes of approved oral antidiabetic drugs. However,
tyrosine phosphatase 1B (PTP-1B). PTP-1B is an enzyme
most of them exert undesirable side effects, drug
interactions and the treatment is expensive since
antidiabetic therapy involve polypharmacy regimen.
located on the cytoplasmic face of the endoplasmic
reticulum, it regulates the target site of insulin in the liver,
muscle and adipose tissue, by dephosphorylating the
activated insulin receptor (IR) and insulin receptor
substrates (IRS). Also, PTP-1B acts as a negative regulator
6
_
_______________
Taken in part from the B. Sc. Pharm. thesis of L. Cerón-Romero
Corresponding author. Tel.: (+52) 777 329 7089
E-mail address: enoch@uaem.mx (S. Estrada-Soto)
1
1, 12
*
in leptin signaling pathway.
Furthermore, in vivo and
1

Scheme 1. Reagents and conditions: (a) mCPBA, CH
2 2
Cl , 25C, MW, 2 h,
in vitro studies showed that PTP-1B inhibition improves
7,13,14
51% yield; (b) K CO , CHCl /DMF, RX, 60 C, MW, 1.5 h, 52-66% yield;
2
3
3
insulin sensitivity and glucose uptake.
Thus, this
2 2 2
(c) DCC, DMAP, PhNH , CH Cl , 55 C, MW, 1 h, 23% yield; (d) Ethylene
enzyme has become in a novel molecular target for the
treatment of diabetes.
4
glycol, PPTS, THF, 55 C MW, 6 h, 23% yield; (e) LiAlH , THF, reflux, 6 h,
50% yield.
To test the ability of each derivative to inhibit
PTP-1B, aliquots of stock solutions (dissolved in DMSO to
prepare 20 mM) were diluted with the assay buffer,
containing 2.5 mM of p-nitrophenyl phosphate (pNPP), to
obtain one inhibitor concentration. Samples were incubated
at 37 °C, and the reactions were initiated by adding an
appropriate enzyme aliquot. The results are summarized in
Figure 2, which shows that all derivatives induced enzyme
inhibition at 50 µM.
120
100
80
60
40
Figure 1. Chemical structure of moronic acid derivatives
20
A series of six moronic acid derivatives were
semisynthesized and examined for their in vitro, in silico
and in vivo antidiabetic effects. Moronic acid (MA,
starting material) was purified from the acetone extract of
0
O
1
2
3
4
5
6
MS
D
the
leaves
and
stems
from
Phoradendron
Figure 2. In vitro assay of the PTP-1B inhibition activity of the moronic acid
derivatives (1-6) at 50 µM. All experiments were performed in triplicate. The
data reported represent the mean±S.E.M. Effect of 4 was not determined.
10
reichenbachianum (Viscaceae) as previously reported.
Six derivatives of MA were prepared (Figure 1): an epoxy-
lactone (1), three C28 esters (2-4), an amide derivative (5)
and an alcohol with the C-3 ketone group protected with
ethylene glycol (6). Scheme 1 depicts the general synthetic
route for the preparation of these compounds. All the
products were obtained under conventional conditions or
microwave-assisted reactions on a CEM Discovery
BechMate system with a potency of 100 W. The
temperature was established for each reaction under open
vessel system. For compound 1, the MA was reacted with
Results indicate that the most active compounds
from the entire series were 2 and 6, showing almost 100%
of enzyme activity inhibition at concentration evaluated.
Therefore, it was decided to determine the IC50 values of
both compounds over PTP-1B. The enzyme activity was
measured at a fixed substrate concentration (corresponding
to the Km) and varying inhibitor concentrations Figure 3.
mCPBA in CH
2
Cl
2
in a 51% yield under MW condition.
A
1.2
1.0
0.8
All esters 2-4 were prepared by classical methods, using
acyl chlorides or alkyl halides, in presence of an
appropriate base, with 52-66% yielding. The reduction
product (6) was obtained with LiAlH
4
in a 50% yield.
0.6
Compound 5 was found in a 23% yield by classical
methods using DCC/DMAP and benzyl amine. Detailed
synthetic procedures and structural characterization for all
these compounds are described at supporting
0
0
0
.4
.2
.0
1
5
information.
1E-3
0.01
0.1
1
10
100
[
2] M
2

buffer. These results can be affected by the fact that 6
showed poor solubility, even in DMSO.
B
1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
The enzyme selectivity of derivatives was
evaluated determining the IC50 values on other PTPs, such
as human TC-PTP, and the isoenzymes IF1 and IF2 of the
human LMW-PTP. As indicated above, results reported in
Table 1 show that, both 2 and 6 displayed quite good
selectivity for PTP-1B respect to the other assayed PTPs.
In particular, 2 shows a strong selectivity for PTP-1B than
TC-PTP, and also good selectivity against both IF1 and IF2
isoenzymes. Furthermore, it is interesting to observe that 6,
despite behaves as a partially reversible inhibitor, displays
good selectivity for PTP-1B compared to human TC-PTP,
and is about 2- and 4-fold selective if its IC50 value is
compared with those on IF1 and IF2 isoenzymes,
respectively.
.0
1
E-3
0.01
0.1
1
10
100
[
6] M
Figures 3. The IC50 values for derivates 2 (A) and 6 (B) were determined by
plotting the data relative to the residual activity of the PTP1B against the
inhibitor concentration. 19 different inhibitor concentrations were used for
each test samples. All tests were performed in triplicate. Data represent the
means ± S.E.M.
Table 1. IC50 values obtained using PTP-1B and other PTPs. For each
inhibitor, between 15 and 18 different inhibitor concentrations were used. All
tests were performed in triplicate. The data represent the mean ± S.E.M
The IC50 values were calculated by fitting
experimental data, using a nonlinear fitting program
FigSys, Biosoft, UK). Results showed in Figure 3 indicate
that the derivative 6 (IC50= 7.5 ± 0.1 M) is more potent
than 2 (IC50= 10.8 ± 0.5 M), which corroborate that the
presence of the carboxylic acid and/or its corresponding
Derivatives
IC50 (M)
TC-PTP
(
PTP-1B
IF1
IF2
2
6
10.8 ± 0.5
> 100
> 100
33.7 ± 1.1 62.5 ± 1.0
16.1 ± 0.4 29.3 ± 0.6
7.5 ± 0.1
carbinol reduction product (H-bond acceptor/ donor) in C-
8
28 is required to maintain the in vitro inhibitory activity.
Based on results and effects described above for
compound 2 which is selective and reversible inhibitor of
PTP-1B, it was decide to define its inhibition mechanism
by the study of the dependence of main kinetic parameters
Also, both compounds were more potent than moronic acid
13.2  0.7), which was used as precursor to synthesized 2
(
7
and 6.
m
(K and Vmax) from inhibitor concentration. Results
To highlight the inhibition mechanism,
obtained were reported in the Figure 5. The straight lines
obtained using differing inhibitor concentrations intersect
in the same point on the Y-axis, demonstrating that 2
behave as competitive inhibitor with respect PTP-1B
enzyme. Also, its experimental Ki was calculated as 3.1 ±
0.2 M, based on eq.1, which indicates that 2 induced a
potent PTP-1B enzyme inhibition (Figure 6). See
reversibility assays were performed. PTP-1B was
incubated in the presence of saturating concentration of
each inhibitor for 90 minutes at 37 °C; then aliquot of
samples were withdrawn and diluted 400-fold with the
assay buffer, and the residual activity of enzyme was
measured. Control assays were carried out adding DMSO,
the solvent used to dissolve derivatives, instead of
inhibitors. Results obtained are reported in Figure 4.
15
Supplementary data.
1
20
00
Km
Kmapp


I  Km

Ki
eq.1
1
8
6
4
2
0
0
0
0
0
50
40
30
20
10
0
DMSO
2
6
Figure 4. Reversibility assay. Aliquot of PTP-1B was incubated in the
presence of DMSO (control) or in the presence of each derivative. In each
samples, the final concentration of derivatives was 10-fold respect to the
concentration corresponding to the IC50 values. After 90 minutes incubation
at 37°C, aliquots of samples were withdrawn and diluted 400-fold with the
assay buffer to determine PTP-1B residual activity. All assays were carried
out in triplicate. The values shown in the figure are the mean ±S.E.M.
-0.5
0.0
0.5
1.0
1.5
1
2.0
-
1
/[pNPP] mM
Figure 5. Lineweaver-Burk plot obtained using PTP-1B enzyme and 2
derivative. The inhibitor concentrations were: , 0 μM; , 2.5 μM; , 5
μM; , 10 μM. All tests were carried out in triplicate. The data reported in
the figure represent the mean ± S.E.M.
Among analyzed derivatives, only 2 behave as a
true reversible inhibitor. In fact, the activity of 6 was
recovered only partially after dilution with the assay
3

1
1
1
4
2
0
8
6
4
2
6
-4
-2
0
2
4
6
8
10
12
[
2] M
Figure 6. Secondary plot showing the dependence of Km from
2
concentration.
In order to gain insight into the putative binding
B
mode of 2 and 6, both compounds were docked with a
1
6,17
crystallographic structure of human PTP-1B,
obtained
from the Protein Data Bank (PDB) accession code 1C83.
Before docking the triterpenic acid derivatives, the
protocol was validated by predicting the binding mode of
the
carboxilic acid, a competitive inhibitor of PTP-1B. Figure
shows a comparison between binding mode of the
crystallographic
6-(oxalyl-amino)-1H-indole-5-
7
crystallographic ligand and the binding mode predicted by
AutoDock 4.2. Figure 7 clearly shows that AutoDock
successfully
crystallographic ligand with a root-mean square deviation
RMSD) of 0.33 Å.
predicted
the
binding
mode
of
(
Figure 8. Docking of the triterpenic acid derivative 2 with PTP-1B. A) 3D
binding mode of tritepenic acid 2. B) Two-dimensional interaction diagram of
the predicted binding mode of 2.
Compounds 6 (Figure 9A and 9B) showed polar
interaction with Gln262 and Van der Waals with Val49,
Ile219, Gly259, Arg254, Ala27, Phe52, Met258, Asp48
and Phe182 with a predictive docking score of -6.78
Kcal/mol, suggesting that the potential binding site could
be localized in an extended binding pocket (site B)
1
6,17
adjacent to the catalytic site A.
The fact that both
compounds 2 and 6 showed interactions with extended
binding site B, could explain the selective inhibition by
PTP-1B instead other phosphatases evaluated in the current
investigation.
A
6
Figure 7. Surface representation of the binding sites of PTP-1B. Catalytic
binding site or site A, is shown in blue; extended binding site B is in red. A
comparison between the binding position of the co-crystal ligand (yellow)
and the binding position predicted by AutoDock (pink) is also shown
(RMSD= 0.33 Å).
Docking score of derivatives with PTP-1B active
site A and extended binding site B, showed that 2 (Figures
A and 8B) had polar and Van der Waals interactions in
8
both sites with Val49, Gln262, Met258, Phe182, Ala217,
Ile219 and Gly259 showing a docking score value of -7.21
16,17
Kcal/mol, being more specific for site A
that explain
the reversible and competitive inhibition described in the
experimental enzymatic inhibition.
A
B
4

40
Vehicle
20
Glibenclamide
6
2
0
20
40
60
-
-
-
*
*
0
1
3
5
7
Hours
* p < 0.05
Figure 10. Effect of a single dose of 2 and 6 (50 mg/Kg; intragastric, n= 5) in
non insulin dependent diabetic rat model. p<0.05 versus vehicle group.
Figure 9. Docking of the triterpenic acid derivative 6 with PTP-1B. A) 3D
binding mode of action of triterpenic acid 6. B) Two-dimensional interaction
diagram of the predicted binding mode of 6.
In particular, derivative 2 showed the best activity,
compared with 6 and control group, producing a sustained
decrease of blood glucose levels, close to 40%, 7 h after
compound administration. It is important to mention that 2
could be act as prodrug of moronic acid since it showed in
vivo significant antidiabetic action after 3 hours post-
treatment, time necessary to metabolize the methyl ester to
the corresponding carboxylic acid.
Finally, as shown compounds 2 and 6 were the
most potent and selective inhibitors of PTP-1B, which
suggest that them could act as in vivo antidiabetic agents.
In this context, both compounds were evaluated in
1
8
experimental non-insulin diabetic rat model.
See
15
Supplementary data. Figure 10 shows that intragastric
administration of 2 and 6 (50 mg/Kg) induced a significant
decrease in plasma glucose concentration in diabetic rats
In summary, compounds 2 and 6 have been
developed as promising bioactive compounds for the
treatment of diabetes. Both compounds exhibited potent
and selectivity inhibition for PTP-1B enzyme, showing
significant oral antidiabetic efficacy.
(
p<0.05) during acute time periods (7 h) compared with the
control group (vehicle).
8
.
Ramírez-Espinosa, J.J.; Rios, M.Y.; Paoli, P.; Flores-Morales, V.;
Camici, G.; de la Rosa-Lugo, V.; Hidalgo-Figueroa, S.; Navarrete-
Vázquez, G.; Estrada-Soto, S.; Eur. J. Med. Chem. 2014, 87, 316.
Rios, M.Y.; López-Martínez, S.; López-Vallejo, F.; Medina-
Franco, J.L.; Villalobos-Molina, R.; Ibarra-Barajas, M.;
Navarrete-Vazquez, G.; Hidalgo-Figueroa, S.; Hernández-Abreu,
O.; Estrada-Soto, S. Fitoterapia. 2012, 83, 1023.
Acknowledgement
9
.
This work was supported in part by Facultad de Farmacia
(
UAEM) internal grants, as well as from SEP-CONACyT
Ciencia Básica (grants 167044 and 241044).
1
1
0. Ramírez-Espinosa, J.J.; García-Jiménez, S.; Ríos M.Y.; Medina-
Franco, J.L.; López-Vallejo, F.; Webster, S.; Binnie M.; Ibarra-
Barajas, M.; Ortiz-Andrade, R.; Estrada-Soto. S. Phytomedicine.
Supplementary Material
2
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Supplementary data associated with this article can be found, in the
online version.
12. Bettaieb, A.; Bakke, J.; Nagata, N.; Matsuo, K.; Xi, Y.; Liu, S.;
AbouBechara, D.; Melhem, R.; Stanhope, K.; Cummings, B.;
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5

Graphical
Abstract
In vitro and in silico PTP-1B inhibition and
in vivo antidiabetic activity of semisynthetic
moronic acid derivatives
Leave this area blank for abstract info.
a
b
b
c
d
Litzia Cerón-Romero, Paolo Paoli, Guido Camici, Virginia Flores-Morales, María Yolanda Rios, Juan J.
a
a
a
a,*
Ramírez-Espinosa, Sergio Hidalgo-Figueroa, Gabriel Navarrete-Vázquez, Samuel Estrada-Soto
40
Vehicle
20
Glibenclamide
6
2
0
20
40
60
-
-
-
*
*
0
1
3
5
7
Hours
* p < 0.05